Lyme disease, caused by the arthropod-borne spirochete Borrelia burgdorferi, is a multisystem disorder that can lead to dermatologic, cardiac, neurologic, and rheumatologic manifestations. With more than 15,000 cases reported in the United States every year, a safe and effective vaccine for preventing Lyme disease has become a public health priority. In this regard, numerous lines of investigation indicate that outer membrane lipoproteins of this organism can be used as effective vaccine reagents. In fact, outer surface lipoprotein A (OspA) has been shown to protect animals and humans from infection with B. burgdorferi, and was approved for use in humans several years ago. However, recent events have led to the OspA vaccine being taken off the market and it is now no longer available to the general public. Therefore, a major emphasis in B. burgdorferi research has been to develop a new vaccine that could be used as a safe and effective second-generation preventative against Lyme disease. Since B. burgdorferi is an extracellular pathogen, and humoral immunity has been shown to be protective against this organism, vaccine studies have revolved around identifying a borrelial antigen that is (i) surface-exposed, (ii) conserved among different strains and genospecies of Borrelia spirochetes, and (iii) expressed during tick transmission and mammalian infection. We recently used microarray experiments to help identify new candidate vaccine molecules that we have shown to be surface-exposed in preliminary studies, designated BbA36, BbA69, and BbI42. Supporting the fact that these antigens may be possible 2nd generation Lyme disease vaccinogens is the fact that antibodies generated against all three can kill B. burgdorferi during in vitro cultivation. Therefore, the major goal of this focused application is to further characterize these surface-exposed proteins by (i) determining their cellular location throughout the enzootic life-cycle of B. burgdorferi, (ii) examining their overall genetic conservation among different strains and genospecies of B. burgdorferi, and (iii) assessing their ability to protect mice and non-human primates from experimental Lyme disease. The proposed studies should lead to candidate vaccine molecules that can be further tested for safety and efficacy in humans for use as a second- generation Lyme disease vaccine.